Thioredoxin h5 is required for victorin sensitivity mediated by a CC-NBS-LRR gene in Arabidopsis

Abstract

The fungus Cochliobolus victoriae causes Victoria blight of oats (Avena sativa) and is pathogenic due to its production of victorin, which induces programmed cell death in sensitive plants. Victorin sensitivity has been identified in Arabidopsis thaliana and is conferred by the dominant gene LOCUS ORCHESTRATING VICTORIN EFFECTS1 (LOV1), which encodes a coiled-coil-nucleotide binding site-leucine-rich repeat protein. We isolated 63 victorin-insensitive mutants, including 59 lov1 mutants and four locus of insensitivity to victorin1 (liv1) mutants. The LIV1 gene encodes thioredoxin h5 (ATTRX5), a member of a large family of disulfide oxidoreductases. To date, very few plant thioredoxins have been assigned specific, nonredundant functions. We found that the victorin response was highly specific to ATTRX5, as the closely related ATTRX3 could only partially compensate for loss of ATTRX5, even when overexpressed. We also created chimeric ATTRX5/ATTRX3 proteins, which identified the central portion of the protein as important for conferring specificity to ATTRX5. Furthermore, we found that ATTRX5, but not ATTRX3, is highly induced in sensitive Arabidopsis following victorin treatment. Finally, we determined that only the first of the two active-site Cys residues in ATTRX5 is required for the response to victorin, suggesting that ATTRX5 function in the victorin pathway involves an atypical mechanism of action.

Figure 1.

Response of Victorin-Insensitive Mutants to Toxin Exposure or Pathogen Infection. (A) Leaves from wild-type LOV1, lov1-6, and liv1-1 plants photographed 3 d after treatment with victorin (top) or 7 d after inoculation with C. victoriae (bottom). n ≥ 50 leaves per line for infection assays. (B) Leaves from wild-type Col-LOV or liv1-4 plants photographed 20 h after infiltration with virulent Pst or Pst carrying avrRpm1 or avrRpt2. n ≥ 48 leaves per treatment per plant line. (C) Leaves from wild-type LOV1 or liv1-1 plants photographed 6 d after infiltration with 5% methanol (left), 100 nM coronatine (top right), or 20 μM fumonisin (bottom right). The underside of the leaves in the top row was photographed to show accumulation of anthocyanin. n ≥ 30 leaves per treatment per line.

Figure 2.

Map-Based Cloning of LIV1. LIV1 was mapped to a 50-kb region flanked by the SSLP markers 17.0ssr3 and 17.1ssr1. Marker positions are indicated in kilobases from the north end of Chromosome I. The relative positions of the subclones spanning this region and the structure of the ATTRX5 gene are shown. Note the orientation of ATTRX5 is reversed with respect to the diagram of the LIV1 region. Arrows indicate positions of EMS-generated point mutations. The attrx5-4 allele is a SALK T-DNA insertion allele.

Figure 3.

Victorin Sensitivity Phenotypes of Plants Expressing Wild-Type or Mutant ATTRX5, Overexpressing ATTRX3, or Mutant for the NADPH-Dependent TRX Reductase Genes NTRA and NTRB. Detached leaves from indicated plant genotypes were treated with 10 μg/mL victorin or water. For (A), (C), and (D), at least eight plants from each of eight T1 lines (64 plants total) were scored for sensitivity to victorin. (A) Leaves from wild-type LOV1 plants, liv1-1 mutant plants, or liv1-1 T1 transgenics transformed with a genomic clone of ATTRX5 photographed 2 d after treatment with victorin. (B) Leaves from plants carrying the LOV1 gene and mutant for either ntra, ntrb, or both photographed 2 d after treatment with victorin. n ≥ 20 leaves per genotype. (C) Leaves from T1 transgenics of liv1-1 plants transformed with 35S:ATTRX5, 35S:ATTRX5(C42S), 35S:ATTRX5(C39S), or 35S:ATTRX5(C39S/C42S) constructs photographed 3 d after treatment with water or victorin. (D) Leaves from wild-type LOV1 plants or liv1-1 T1 transgenics transformed with a 35S:ATTRX3 construct photographed 2 d after treatment with victorin.

Figure 4.

Correlation of ATTRX3 Transgene Expression with Victorin Sensitivity. Twenty 35S:ATTRX3 T0 transgenics were scored for victorin sensitivity by the detached leaf assay. Six of these plants were evaluated for level of transgene expression by RNA gel blot analysis. Note that this blot was exposed for a very short time to allow visualization of differences in band intensities between lanes. Ethidium bromide staining of the RNA gel is shown to confirm equal sample loading.

Figure 5.

RNA Gel Blot Analysis of ATTRX5 and ATTRX3 Gene Expression. ³²P-labeled probes were used to monitor expression of ATTRX5 and ATTRX3 in the indicated plant genotypes. Ethidium bromide staining of the RNA gels is also shown to confirm equal sample loading. Time points are given in hours after infiltration with 30 μg/mL victorin. The first lane of each gel contains RNA from untreated leaves (U). (A) ATTRX5 and ATTRX3 expression in victorin-sensitive plants from the LOV1 line. (B) ATTRX5 and ATTRX3 expression in victorin-insensitive lov1-6 mutant plants. (C) ATTRX5 and ATTRX3 expression in victorin-insensitive liv1-1 mutant plants. (D) ATTRX5 and ATTRX3 expression in victorin-sensitive plants from the Col-LOV line. (E) ATTRX5 expression in plants homozygous for LOV1 and for the indicated defense response mutant allele.

Figure 6.

Construction and Evaluation of ATTRX5/ATTRX3 Fusion Constructs. (A) Alignment of ATTRX5 and ATTRX3 amino acid sequence by ClustalW. Conservative amino acid substitutions are highlighted in gray. Active-site residues are denoted by asterisks. Arrowheads show location of restriction sites used to splice the ATTRX5 and ATTRX3 coding sequences for the indicated constructs. (B) Diagram showing the portions of ATTRX5 and ATTRX3 coding sequences present in each construct. Numbers to the immediate right indicate the number of amino acid substitutions relative to wild-type ATTRX5, and the numbers in parentheses indicate the number of nonconservative substitutions relative to wild-type ATTRX5. (C) Representative leaves illustrating the symptom rating scale used to evaluate the degree of victorin sensitivity conferred by each construct.